Ever since my undergraduate years in biochemistry, I have
developed an intense fascination with the chemistry and biology of membranes. While
most elementary textbooks on biology have often portrayed the cell membrane as
a pair of lines (or curves in some cases), this organelle is actually one of
the most dynamic components of the cell, and the chemistry and molecular
biology behind it serve as the driving force for many wonderful molecular
phenomena.
Now, I am working in the field of bi-phasic catalysis, and it is
quite gratifying to see that there are parallels between my field and that of
membrane science. If possible in the future, membrane chemical biology is
certainly a research field I would love to explore! This week, I have read a
great article in Journal of American Chemical Society about lipid membrane and
hydrogel formation [1], and I would like to share with you here.
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| Figure 1. Hydrogel network formation through the catalysis of a lipid membrane. |
The paper is about the use of a negative-charged lipid
membrane as a catalyst to facilitate the formation of a hydrogel network (Figure 1). A
hydrogel is defined as a polymeric, gel-like macromolecular structure, which is
made by the cross-linking of polymer chains. Hydrogels have been used in drug
delivery, tissue engineering, supramolecular catalysis [2c], and many other
fields. The self-assembly and
aggregation of the resulting fibrous hydrogel network has to be designed in a
way so that the material properties of the resulting gel fiber can be
controlled. On the other hand, the spatial position of hydrogel formation has
to be carefully defined, to serve the aim of controlled release or delivery of,
for example, drug molecules.
The researchers have found that a negatively-charged lipid
membrane can be used as a catalyst to form supramolecular hydrogel networks. The
negatively-charged liposomes can be used to catalyze the formation of a gelator
molecule 3, and they have been able
to achieve spatial control – the gelator molecule 3 is formed near the membrane, so that the resulting hydrogel
networks can be formed in this well-defined area. The mechanism involves the
generation of a local proton (H+) gradient - due to the prevalence
of the negative charges. The high proton concentration facilitates the
acid-catalyzed formation of hydrazone– the
functional group in the gelator molecule 3.
For the chemistry, the first stage to form the hydrogel is a
reaction between a hydrazide 1 and
an aldehyde 2 to form the hydrazone
derivative 3. At neutral pH, this
reaction is very slow. Yet, the researchers have established that, when an acid
or aniline is added, the reaction rate improves a lot. The structure of the
hydrazone enables itself to self-assemble and forms a fiber-like structure, and
aggregates towards a cross-linked network, resulting in gelation of the
surrounding solvent.
The researchers are interested to see whether a negatively-charged
lipid membrane can somehow catalyze the formation of hydrazone 3 and also the formation of the final
hydrogel network. Their rationale is that the negative charges on the membrane
can induce an increase in the proton concentration, leading to a decrease of pH
and renders the chemical environment more acidic. They believe this acidic
environment may catalyze the formation of the hydrazone stucture and subsequent
hydrogelation.
The reaction is carried out in the following way. The hydrazide
1 and the aldehyde 2 are mixed in a defined ratio in a
buffered solution at neutral pH. After that, the negative-charged liposome
solution is added, and the reaction is carried out at room condition. As
observed, the gelator molecule 3 is
formed and eventually gelation of solvent occurs, signifying the complete formation
of the hydrogel network.
How did the researchers access whether catalysis occurred from
the liposome? They employed a parameter known as minimum gelation concentration
(MGC). They have first measured a control value – where no liposome is added to
3 at pH 7. When a liposome from a lipid
with negative charges, DPPG (Figure 2), is added to the reaction mixture with 1 and 2, a decrease in MGC is evident. Of course, if an acid or aniline
is added to the reaction mixture instead, due to their catalytic effects, the
MGC should also decrease. Indeed, the researchers have found that the negatively
charged liposomes work even better – they lead to lower MGC as compared to the
acid / aniline scenarios. As another control experiment, the addition of a positive
charged liposome sample does not lead to a decrease of MGC. With the use of an
UV-active hydrazide substrate, the researchers can monitor the formation of
hydrazone using absorbance spectroscopy for different reaction conditions. Thus,
the logical conclusion from these trials is that a negative charged membrane is
essential for catalysis to occur.
 |
| Figure 2. Structure of DPPG and DOPG. |
Yet, there is one important thing to note before we jump to
simple conclusions. The researchers have found that, while negatively charged
membrane is likely to catalyze the formation of hydrogelator network, not all negative charged membranes can achieve that. The one missing piece in the
puzzle is the melting temperature, Tm, of the liposome. When the
researchers added the liposome from the negative charged lipid DOPG (Figure 2, Tm
-20oC, a liquid phase membrane at room temperature), to the reaction
mixture, no hydrogelation occurs even at high liposome concentration. Yet, when
the researchers increase the rigidity of the DOPG membrane through the addition
of cholesterol (this should be familiar to anyone doing biochemistry or
membrane biology), the DOPG-cholesterol hybrid membrane can then catalyze
hydrogelation, suggesting the Tm of this hybrid liposome has
increased.
Thus, the researchers have summarized that, in order for the
liposome to catalyze hydrogelation, 2 criteria have to be fulfilled:
(1)
a negatively charged membrane surface
(2)
a solid phase at room temperature
The researchers have also established, from oscillatory
rheology, that lipid concentration can control the physical properties of the hydrogel
network. They also believe that liposomes are serving as nucleation points for
the formation of fibrous network. I can think of a similar analogy in the case
of cytoskeleton biology, where accessory proteins can serve as nucleation
centers for actin polymerization.
By using confocal microscopy and a fluorescent aldehyde substrate,
the researchers can also visualize the formation of the hydrogel network. When
no liposomes are present, the resulting structure is very slack and
un-connected. In the presence of liposomes, by contrast, the resulting hydrogel
network becomes well-organized and dense, and the effect is enhanced when the
liposome concentration is increased.
A very interesting aspect of the gel fiber formation occurs
from the ‘underdog’, DOPG, which does not meet up to potential at the catalytic
tests only until cholesterol comes to help. Rather curiously, because the Tm
of DOPG is low, that meant the membrane it forms is more fluid than the
membrane from DPPG. If we look at the chemical structure of DOPG and DPPG, it
may shed light on this observation, and this concept is also familiar to
biochemistry students. DOPG contains carbon-carbon double bonds, while DPPG is
totally saturated on the carbon chains. The presence of double bonds will
provide kinks and prevent a close-packing of the hydrocarbon chains, which by
contrast is facile when only saturated chains are present. Thus, the
unsaturated DOPG is more fluid than the saturated DPPG, and this also explains
why the Tm of DOPG is lower.
DOPG has a higher affinity for the gel fibers. The affinity
of the hydrogel fiber for the lipid membrane is related to the phase behavior
of the hydrocarbon chains of the lipids, thus, a more fluid membrane should favor
this interaction. Thus, DOPG-derived membrane seems to interact better with the
hydrogel fiber than DPPG-type membrane.
What I am particularly impressed is that, by carrying out so
many control experiments, the researchers draw together all the clues and
provide a coherent explanation for the different performance of the DPPG and
DOPG in catalysis. They propose that, because the DPPG membrane has less
affinity to the hydrogel fiber, so the fiber is not blocking the way for DPPG
to effect catalysis on its surface, therefore an efficient catalysis occurs and
it goes on and on. In contrast, DOPG-type membrane, which is fluid and ‘loves’
the hydrogel fiber, interacts with the
gel fiber with such a high affinity that it is literally blocking the way for
further rounds of catalysis. The researchers also draw analogy to the product
blocking phenomena in heterogeneous catalytic systems, and I find this as an
impressive explanation!
At the biological side, the researchers have also generated hydrogel
fiber formations on HeLa cell systems.
All in all, this is a wonderful paper on membrane chemical
biology. I have learnt a number of new techniques from it, and I am
particularly impressed by the mechanistic insights, both in terms of catalytic
and material, from all the great experiments they have carried out to arrive at
the conclusions. Brilliant!
by Ed Law
13/7/2016
Reference:
1. Negatively Charged Lipid Membranes Catalyze Supramolecular
Hydrogel Formation
Frank Versluis, Daphne M. van Elsland, Serhii Mytnyk,
Dayinta L. Perrier, Fanny Trausel, Jos M. Poolman, Chandan Maity, Vincent A. A.
le Sage, Sander I. van Kasteren, Jan H. van Esch and Rienk Eelkema
J. Am. Chem. Soc., 2016, asap, DOI: 10.1021/jacs.6b03853
2. Originally, I plan to talk about a self-emulsifying
system which is used in the hydroformylation of lipid substrates. When I read
Ref. [1], I decide to talk about that instead. It is interesting to see there
are some connections regarding the two topics. Here are the references:
(a) A self-emulsifying catalytic system for the aqueous
biphasic hydroformylation of triglycerides
T. Vanbésien, A. Sayede,
E. Monflier and F. Hapiot
Catal. Sci. Technol., 2016,6, 3064-3073
DOI: 10.1039/C5CY01758K
(b) Supramolecular Emulsifiers in Biphasic Catalysis: The
Substrate
Drives Its Own Transformation
Théodore Vanbésien, Eric Monflier, and Frédéric Hapiot
ACS Catal. 2015, 5, 4288−4292
DOI: 10.1021/acscatal.5b00861
(c) Thermoresponsive Hydrogels in Catalysis
Frédéric Hapiot, Stéphane Menuel, and Eric Monflier
ACS Catal. 2013, 3, 1006−1010
dx.doi.org/10.1021/cs400118c
